Water soluble zanthylium derivative substrates

ABSTRACT

Water soluble xanthylium derivative substrates of rhodamine 110 and rhodol permit spectrophotometric and fluorescent measurements of trypsin-like enzymes without the addition of organic solvent additives and/or special water solubilizing agents. These novel substrates exhibit increased sensitivity for determining low levels of activity of trypsin-like enzymes such as proteolytic enzymes, cofactors, activators, antiactivators, and inhibitors. These substrates can be substituted for fibrinogen to monitor the pathways of blood coagulation.

This application is a division of application Ser. No. 737,761, filedMay 28, 1985 now U.S. Pat. No. 4,694,070.

FIELD OF THE INVENTION

This invention relates to water soluble xanthylium derivativesubstrates, method of their synthesis, and utilization of such syntheticsubstrates for measurement of the activity of protease enzymes,inhibitors, cofactors, activators, and antiactivators, individually orin combination with each other.

BACKGROUND OF THE INVENTION

Synthetic rhodamine 110 derivative substrates heretofore have beenutilized for fluorometric measurement of protease enzyme activity, asreported by Leytus et al., Biochem. Journal 209: 299-307 (1983) andLeytus et al., Biochem. Journal 215: 253-260 (1983). The Leytus et al.,substrates were extremely sensitive for measurement of enzymeactivities, as compared to previously reported substrates. However, theLeytus et al. rhodamine 110 derivative substrates have been found toexhibit low water solubility of about 120 micromolar at 37° C., therebynecessitating the addition of organic solvent additives, e.g.dimethylformamide and ethanol, for promoting the substrate solubility.However, addition of organic solvents to enzyme assays is known tointerfere with the assay procedure, i.e. denaturing the protein,decreasing reaction rate and/or causing precipitation of the otherreactants or products, resulting in less than optimum test performance.

In addition to the disclosure of Leytus et al., a coumarin derivativesubstrate for the fluorometric and spectrophotometric determination ofproteolytic enzymes in biological fluids was disclosed by Smith et al.,U.S. Pat. No. 4,294,923. The Gargiulo et al. U.S. Pat. Nos. 4,275,153and 4,336,186 disclose a fluorogenic aminoisophalate substrate fordetermination of protease enzyme activities in biological samples.

SUMMARY OF THE INVENTION

The present invention relates to water soluble xanthylium derivativesubstrates, hereinafter referred to as rhodamine 110 and rhodol, forfluorometric and spectrophotometric measurement of protease enzymeactivities which, because of their water solubility at workingconcentrations, obviate the necessity of using organic solubilizingagents, e.g. dimethylformamide and ethanol, and thus eliminates theirconcomitant test interferences. In addition, the water soluble rhodamine110 and rhodol derivative substrates exhibit greater sensitivity fordetermining levels of enzyme activities than the rhodamine 110derivative substrates of the prior art. Consequently, as a result ofthis increased sensitivity, the water soluble rhodamine 110 and rhodolderivative substrates have utility in spectrophotometric testprocedures; whereas, the prior art rhodamine 110 derivative substratesof low water solubility do not exhibit the sensitivity sufficient to beused with smaller volumes of sample and substrate to obtain bothspectrophotometric and fluorescent determinations. The water solublexanthylium derivative substrates of the present invention are useful tomeasure the activity of protease enzymes, cofactors, inhibitors,activators, and antiactivators, individually or in combination with eachother, and specifically to measure the activity of trypsin-like enzymes.

An object of this invention is to provide a water soluble complex ofxanthylium derivative substrates with side chains which are hydrolyzedby the enzymes, such that the hydrolysis of the side chains is directlyproportional to the enzyme activity.

Another object of this invention relates to providing xanthyliumderivative substrates with increased water solubility.

A further object of this invention, as compared to the prior art, is toeliminate the need for organic solvents in the assay procedures formeasuring the activity of protease enzymes, cofactors, inhibitors,activators, and antiactivators, and specifically thrombin formation.

A still further object of this invention is to provide xanthyliumderivative substrates with sufficient sensitivity to performspectrophotometric, as well as fluorometric, measurements.

PREFERRED EMBODIMENTS

The synthetic substrates herein and their hydrolysis products are watersoluble at working concentrations, thereby permitting their use for bothspectrophotometric and fluorescent measurements, without the use oforganic solvent additives and/or special water solubilizing agents. Thenovel water soluble substrates have the following general formulas:##STR1## wherein

R₁ is a water solubilizing radical selected from the group consisting ofsarcosyl, pyroglutamyl, and glutaryl;

R₂ is xanthylium, 3',6'-diamino-9'-(2-carboxyphenyl); and

R₃ is xanthylium, 3'-amino-6'-hydroxy-9'-(2-carboxyphenyl) and the watersoluble salts thereof.

The chemical structure for rhodamine 110 and rhodol are set forth below:##STR2##

The sensitivity of the protease enzymes for these compounds is increasedto include detection of lower levels of enzyme, inhibitor, cofactor,activator, and antiactivator than previously detectable using knownmethods; sensitivity being defined as the least detectable quantity.These improvements allow assay of thrombin by prothrombin time (PT),activated partial thromboplastin time (APTT) and thrombotest (TT) to beperformed using the novel water soluble rhodamine 110 and rhodolderivative substrates of the present invention.

EXAMPLES

Synthesis of the water soluble xanthylium derivative substrates,analytic test results and examples of use of the substrates aredescribed below. The following examples are not meant to limit the scopeof the invention.

All amino acids used have the L-configuration, except as otherwisestated. The abbreviations used in the following examples are:

Arg=Arginine

Cbz=Carbobenzyloxy

DMF=Dimethylformamide

EDAC=1-Ethyl-3-(3'-Dimethylaminopropyl)-Carbodiimide HCl

Glt=Glutaryl

HAc=Acetic acid

Pro=Proline

Pyro=Pyroglutamyl

Sar=Sarcosyl

In the thin layer chromatography (TLC) analysis of the eluate andproducts, precoated glass plates are used with silica gel 60,254 (EM,Co. reagents) as absorption medium. For the development of the thinlayer chromatograms, the following solvent systems are used:

S₁ =2-butanone:acetone:H₂ O; 8:1:1 (V:V)

S₂ =methanol:acetic acid:H₂ O; 9:0.5:0.5 (V:V)

SYNTHESIS OF THE SYNTHETIC SUBSTRATES

Water soluble xanthylium derivative substrates are synthesized by firstpreparing a xanthene derivative substrate having the formula(H-L-Pro-Arg)₂ -Rhodamine 110. The synthesis of (H-L-Pro-Arg)₂-Rhodamine 110, is fairly taught by Leytus et al.

EXAMPLE I Prior Art

Step 1:

Dissolve 0.96 g (2.62 mmol) of Rhodamine 110 in 400 mL of colddimethylformamide (DMF) in a 1:1 ratio of volume to volume (V:V) withPyridine at ice-bath temperature of 4° C., with concurrent stirring, andthereafter add to the admixture 25.6 g (135.5 mmol) of EDAC. Afterstirring for about one minute, rapidly add a solution of 11.52 g (33.41mmol) of Cbz-L-Arginine-HCl in 96 mL of cold DMF:Pyridine solution in a1:1 ratio. After a period of about 24 hours, the resulting product isisolated by adding 1.6 L of anhydrous ether, followed by centrifugingfor about 10 minutes at 10,000 g, forming an oil-like product. Theresulting oil is dissolved in 50 mL cold DMF and precipitated by theaddition of 1.3 L cold acetone. The oil is separated by centrifugationfor 10 minutes at 10,000 g. The oil is redissolved in 50 mL cold DMF and500 mL cold 1.2N HCl is added. A red precipitate is isolated bycentrifugation for 10 minutes at 10,000 g. The red precipitate isdissolved in 50 mL cold methanol and precipitated by the addition of 350mL cold ethyl acetate. The resulting precipitate, (Cbz-L-Arg)₂-Rhodamine 110, is collected by centrifugation for 10 minutes at 10,000g. The last step is repeated three times.

Yield: 2.08 g. (+79.4%) of (Cbz-L-Arg)₂ -Rhodamine 110.

Step II

Dissolve 3.4 g (3.40 mmol) of (Cbz-L-Arg)₂ -Rhodamine 110, as preparedin Step I, in 75 mL of a 32% HBr-HAc solution. The resulting red coloredsolution is stirred at room temperature (RT) for about 1 hour. Theresulting product is isolated by centrifugation with 800 mL of anhydrousether. After repeating the centrifuging step an additional three times,the resulting brown solid, (H-L-Arg)₂ -Rhodamine 110, is dried in vacuofor 16 hours.

Yield: 3.4 g (+100%) of (H-L-Arg)₂ -Rhodamine 110.

TLC, S₂ : R_(f) 0.09.

Step III

Dissolve 2.75 g (2.79 mmol) of (H-L-Arg)₂ -Rhodamine 110, as prepared inStep II, in 330 mL of a 1:1 DMF:Pyridine volume to volume (V:V) solutionwith concurrent stirring at ice-bath temperature of 4° C., andthereafter add an admixture of 32 g (166.9 mmol) of EDAC. After stirringfor about one minute to accelerate dissolving, rapidly add a solution of10.40 g (41.7 mmol) of Cbz-L-Proline in 60 mL 1:1 DMF:Pyridine solution.After a period of about 24 hours, the product is isolated by theprocedures in Step I. The resulting tan colored solid, (Cbz-L-Pro-Arg)₂-Rhodamine 110, is dried in vacuo for 16 hours.

Yield: 1.86 g (52.0%) of (Cbz-L-Pro-Arg)₂ -Rhodamine 110.

TLC, S₂ : R_(f) 0.44.

Step IV

Dissolve 1.80 g (1.40 mmol) of (Cbz-L-Pro-Arg)₂ -Rhodamine 110, asprepared in Step III, in 45 mL of a 32% HBr-HAc solution, stirring theresulting red colored solution at room temperature (RT) for about 1hour. The resulting product is isolated by centrifugation with anhydrousether, as described in Step I. The resulting brown colored solid,(H-L-Pro-Arg)₂ -Rhodamine 110, is dried in vacuo for 16 hours.

Yield: 1.90 g (+100%) of (H-L-Pro-Arg)₂ -Rhodamine 110.

TLC, S₂ : R_(f) 0.03.

EXAMPLE II Procedure for Producing (H-Sar-Pro-Arg)₂ -Rhodamine 110

Step A

Dissolve 1.90 g (1.63 mmol) of (H-L-Pro-Arg)₂ -Rhodamine 110, asprepared in Steps I-IV, in 190 mL of a 1:1 DMF:Pyridine solution atice-bath temperature of 4° C., and thereafter add to the admixture 17.85g (93.1 mmol) of EDAC. After stirring for about one minute to acceleratedissolving, rapidly add a solution of 5.20 g (23.3 mmol) ofCbz-Sarcosine in 40 mL of a 1:1 DMF:Pyridine solution. After a period ofabout 24 hours, the resulting product is isolated by the proceduresdescribed in Step I. The resulting pink colored solid,(Cbz-Sar-Pro-Arg)₂ -Rhodamine 110, is dried in vacuo for 24 hours.

The yield of (Cbz-Sar-Pro-Arg)₂ -Rhodamine 110 is 1.1 g (49.6%).

Step B

Dissolve 1.1 g (0.78 mmol) of (Cbz-Sar-Pro-Arg)₂ -Rhodamine 110, asprepared in Step A, in 24 mL of a 32% HBr-HAc solution, stirring theresulting deep-red solution at room temperature (RT) for about 1 hour.The resulting product is isolated by centrifugation with 350 mL ofanhydrous ether. After repeating this step an additional three times,the resulting brown colored solid, (H-Sar-Pro-Arg)₂ -Rhodamine 110, isdried in vacuo for 24 hours.

The yield of (H-Sar-Pro-Arg)₂ -Rhodamine 110.51/2 HydrobromidePentahydrate is 1.00 g (84.7%).

Elemental Analysis for C₄₈ H₆₄ N₁₄ O₉.51/2HBr.5H₂ O.

    ______________________________________                                        Theoretical, %        Found, %                                                ______________________________________                                        C = 38.00             37.84, 38.11                                            H = 5.24               5.17, 4.81                                             N = 12.93             12.69, 12.76                                            Br = 29.03            29.58                                                   TLC, S.sub.2 : R.sub.f 0.01.                                                  ______________________________________                                    

EXAMPLE III Procedure for Producing (H-L-Pyro-Pro-Arg)₂ -Rhodamine 110

Step A

Dissolve 380 mg (0.325 mmol) of (H-L-Pro-Arg)₂ -Rhodamine 110, asprepared in Steps I-IV, in 50 mL of a 1:1 DMF:Pyridine solution atice-bath temperature of 4° C., and thereafter add to the admixture 3.58g (18.62 mmol) of EDAC. After stirring for about one minute toaccelerate dissolving, rapidly add a solution of 1.23 g (4.66 mmol) ofCbz-L-Pyroglutamic Acid in 11 mL of cold 1:1 DMF:Pyridine. After aperiod of about 24 hours, the resulting product is isolated by theprocedures as described in Step I. The resulting brown colored solid,(Cbz-L-Pyro-Pro-Arg)₂ -Rhodamine 110, is dried in vacuo for 16 hours.

The yield of (Cbz-L-Pyro-Pro-Arg)₂ -Rhodamine 110 is 278 mg (57.1%).

Step B

Dissolve 78 mg (0.0521 mmol) of (Cbz-L-Pyro-Pro-Arg)₂ -Rhodamine 110 in3 mL of a 32% HBr-HAc solution and stir the resulting red coloredsolution at room temperature (RT) for about 1 hour. The resultingproduct is isolated by centrifugation with 75 mL of anhydrous ether.After repeating this centrifugation step three additional times, theresulting brown colored solid, (H-L-Pyro-Pro-Arg)₂ -Rhodamine 110, isdried in vacuo for 16 hours.

The yield of (H-L-Pyro-Pro-Arg)₂ -Rhodamine 110.41/2 Hydrobromide.51/2Hydrate is 57 mg (71.7%).

Elemental Analysis for C₅₂ H₆₆ N₁₄ O₁₁.41/2HBr.51/2H₂ O.

    ______________________________________                                        Theoretical, %       Found, %                                                 ______________________________________                                        C = 40.90            40.54, 40.32                                             H = 5.01              5.08, 5.06                                              N = 12.85            12.55, 12.73                                             Br = 23.60           23.56                                                    ______________________________________                                    

EXAMPLE IV Process for Producing (Glt-Pro-Arg)₂ -Rhodamine 110

Step A

Dissolve 110 mg (0.094 mmol) of (H-L-Pro-Arg)₂ -Rhodamine 110, asprepared in Steps I-IV, in 12 mL of a 1:1 DMF:Pyridine solution atice-bath temperature of 4° C., and thereafter add to the admixture 171mg (1.5 mmol) of Glutaric anhydride. After a period of about 24 hours,the resulting product is isolated by the procedures as described in StepI. The resulting brown colored solid, (Glt-Pro-Arg)₂ -Rhodamine 110, isdried in vacuo for 16 hours.

The yield of (Glt-Pro-Arg)₂ -Rhodamine 110 is 118.3 mg (97.5%).

EXAMPLE V Process for Producing Cbz-Arg Rhodol

Step A

Dissolve 3.5 g (0.54 mmol) of Rhodamine 110 in 25 mL of concentratedsulfuric acid at room temperature (RT) with concurrent stirring, andthereafter add to the admixture a solution of 700 mg (1.45 mmol) ofsodium nitrite and 10 mL of concentrated sulfuric acid over a period ofabout 20 minutes. After a period of about 16 hours, the resultingadmixture is poured into 250 g of ice-water. The resulting dark redsolution, a diazo compound, is heated to a temperature of 65°-70° C. forabout 30 minutes and nitrogen is evolved. The resulting lighter coloredsolution is filtered without cooling. After cooling, a red-brownflocculent precipitate is filtered, washed with a small amount of waterand dried at 90° C. in vacuo for 16 hours. The red-brown flocculentproduct, Rhodol hemisulfate, appears to be 75% by TLC.

The yield of Rhodol hemisulfate is 3.5 g (96.6%).

TLC, S₁ : R_(f) 0.70.

Step B

Dissolve 1.14 g (3 mmol) of Rhodol hemisulfate in 500 mL of a 1:1DMF:Pyridine solution at ice-bath temperature of 4° C., and thereafteradd to the admixture a solution of 8.98 g (75 mmol) EDAC. After stirringfor about one minute, rapidly add a solution of 6.55 g (19 mmol) ofCbz-L-Arg-HCl in 60 mL of a 1:1 DMF:Pyridine solution. After a period ofabout 24 hours, the resulting product was isolated by the proceduresdescribed in Step I. The resulting reddish brown color solid,Cbz-L-Arg-Rhodol, is dried in vacuo for 16 hours.

The yield of Cbz-L-Arg-Rhodol is 0.2 g. (10%).

TLC, S₁ : R_(f) 0.20.

SOLUBILITY--COMPARISON OF (Sar-Pro-Arg)₂ -RHODAMINE 110 AND(Cbz-Pro-Arg)₂ -RHODAMINE 110 OF THE PRIOR ART

An example of the enhanced sensitivity of the water soluble rhodamine110 derivative substrates is demonstrated by the following data:Thrombin reaction rates were determined with the water solublesubstrate, (Sar-Pro-Arg)₂ -Rhodamine 110 and with the low water solublesubstrate (Cbz-Pro-Arg)₂ -Rhodamine 110 of the prior art. Identical testconditions were used including temperature 37° C., volume 1.0 ml., andbuffer 0.10M ACES pH 7.0, with 0.05M KCl. Since sarcosyl (Sar) andcarbobenzyloxy (Cbz) have similar molecular weights, both were tested at100 ug/ml. The use of 15% ethanol was required to promote the solubilityof the (Cbz-Pro-Arg)₂ -Rhodamine 110. The solubility of the Cbzderivative substrate, even with the addition of 15% ethanol, was up to120 micromolar at 37° C. In contrast, the solubility of (Sar-Pro-Arg)₂-Rhodamine 110 was up to 120 millimolar at 37° C., a thousand-foldincrease in solubility.

    ______________________________________                                        Change In Absorbance, 496 nm/Unit Thrombin/mL                                                   Present Invention                                           Prior Art         (Sar--Pro--Arg).sub.2 --Rhodamine                           (Cbz--Pro--Arg).sub.2 --Rhodamine                                                               110                                                         110                            800                                            80 micromolar     80 micromolar                                                                              micromolar                                     ______________________________________                                        0.122             1.700        2.872                                          ______________________________________                                    

The above data, comparing the prior art substrate to the substrate ofthe present invention, demonstrates that the water soluble derivativesubstrate of the present invention is approximately fourteen times moresensitive than the low water solubility substrate of the prior art atthe same molar concentration. The substrate of the present invention isas much as twenty-four times more sensitive at the higher concentrationof the water soluble substrate than the prior art substrate.

ASSAYS USING THE WATER SOLUBLE XANTHYLIUM DERIVATIVE SUBSTRATES

The novel water soluble xanthylium derivative substrates permitdetermination of the activity of proteolytic enzymes, inhibitors,cofactors, activators, and antiactivators by spectrophotometric andfluorescent determinations. The novel water soluble xanthyliumderivative substrates can be substituted for the natural substrate,fibrinogen, in blood coagulation tests based on the clotting of fibrin.

To more fully understand the invention, several assay examples are nextpresented.

ASSAY EXAMPLES Example I Spectrophotometric Assay of Thrombin with(Pyro-Pro-Arg)₂ -Rhodium 110

Dose response curves were determined for three different concentrationsof thrombin by measuring absorbance increases at 468 nm and 496 nm.Working thrombin dilutions were prepared as follows: Purified humanalpha-thrombin, Dr. John Fenton, N.Y. State Dept. of Health, was dilutedto 1.04 U/mL, 0.52 U/mL and 0.25 U/mL in 0.05M HEPES, pH 8.0, with 0.25MNaCl and maintained at 4° C. The substrate, (Pyro-Pro-Arg)₂ -Rhodamine110, was dissolved to 78 ug/mL in 0.20M HEPES, pH 8.0, with 0.3M KCl.Disposable acrylic cuvettes with 1 cm light path, Centaur Sciences, Inc.were used. A one mL aliquot of substrate was pipetted into a cuvette andwarmed to 37° C. in a heating block. A one mL sample of a workingthrombin dilution was added to the cuvette. After mixing the thrombinand substrate, absorbances at 468 nm and 496 nm were determined for 30seconds with a Hewlett-Packard Model 8450A spectrophotometer. Theincreased absorbances over the timed intervals were:

    ______________________________________                                                     Absorbance, 0-30 seconds                                         Thrombin, U/mL 468 nm     496 nm                                              ______________________________________                                        0.26           0.0461     0.0462                                              0.52           0.0912     0.0860                                              1.04           0.1766     0.1671                                              ______________________________________                                    

A linear relationship exists between the thrombin concentrations and themeasured absorbances at each wavelength:

    ______________________________________                                                           468 nm                                                                              496 nm                                               ______________________________________                                        Correlation Coefficient, r =                                                                       0.9999  0.9996                                           ______________________________________                                    

The relationship between the measured absorbances at 468 nm and 496 nmwas: Correlation Coefficient, r=0.9998. The above data establishes theability of absorbance readings to be measured at either 468 nm or 496nm.

Example II Fluorescent Coagulation Factor Assays with (Sar-Pro-Arg)₂-Rhodamine 110

Fluorescent assays for coagulation factors VIII and IX, based on theactivated partial thromboplastin time (APTT) assay, were performed withthe substrate (Sar-Pro-Arg)₂ -Rhodamine 110. The synthetic substrate wasused to detect thrombin generation, substituting for the naturalsubstrate fibrinogen present in both human and animal plasmas. Standardcalibration curves were constructed with normal plasma, Thromboscreen™from Curtin Matheson Scientific, which was used to correct factor VIIIand IX deficient plasmas, George King Biomedical. The APTT reagentcontained purified soybean phospholipids and ellagic acid activator,Dade Diagnostics. A 125 ug/ml sample of substrate was dissolved in 0.10MACES, pH 7.0, with 0.05M KCl and 5 mM CaCl₂. A Turner model 430spectrofluorometer with a temperature controlled sample cell holder (37°C.) was used to make the kinetic fluorescent measurements. Excitationand emission wavelengths were set at 468 nm and 525 nm, respectively.Glass test tubes were used to perform the plasma activations anddisposable acrylic cuvettes, Centaur Sciences, Inc., were used for thefluorescent measurements.

A 100 uL aliquot of 0.85% saline or normal plasma diluted in saline wasadded to a test tube followed by the sequential addition of 100 uL offactor deficient plasma and 100 uL of the APTT reagent. The test tubewas incubated in a heating block at 37° C. for 2 minutes, at which time100 uL of 25 mM CaCl₂ was added and the incubation was continued for anadditional 30 seconds. The incubation mixture was transferred to acuvette containing 2.0 mL of the substrate solution prewarmed to 37° C.and the fluorescent rate measured. Linear dose response curves wereobtained for factors VIII and IX between 1% and 100% of normal:

    ______________________________________                                        Plasma                  Relative Fluorescence*                                Dilution                                                                             Factor Level, % Normal                                                                         Factor VIII                                                                              Factor IX                                  ______________________________________                                        --     <1               0.119      0.121                                      1:250   1               0.121      0.127                                      1:25    10              0.148      0.154                                      1:2.5  100              0.333      0.333                                      ______________________________________                                         *The relative fluorescence of 0.85 ug quinine sulfate/mL 0.1 N H.sub.2        SO.sub.4 was 1.0 under the same measurement conditions, except for            excitation and emission wavelengths of 350 nm and 450 nm, respectively.  

Example III Spectrophotometric Assay of Plasminogen with (Sar-Pro-Arg)₂-Rhodamine 110

Streptokinase activated plasminogen was measured in plasma samples andthe results were compared to a standard fluorescent assay procedure,Pochron, S. P. et al., Thrombosis Research 13, 733-739 (1978).Lyophilized normal citrated plasma from Hyland Diagnostics was used toprepare the assay standard calibration curves. Streptokinase,Calbiochem-Behring was reconstituted to 2,000 units/mL in 0.02M HEPES,pH 7.5. (Sar-Pro-Arg)₂ -Rhodium 110 was dissolved in 0.10M ACES, pH 7.0,with 0.05M KCl, to 375 ug/mL. The assay was performed with 20 uL eachplasma sample and 50 uL H₂ O added to a semi-micro disposable cuvette, 1cm light path, Evergreen Scientific. After warming to 37° C. in aheating block, 500 uL of the Streptokinase solution was added withmixing and allowed to incubate at 37° C. for 2 minutes 48 seconds. Theactivation time of 2 minutes 48 seconds was used, since it is the fixedtiming increment of a COULTER® DACOS® Chemistry system. Full plasminogenactivation occurs in about 2 minutes, but longer activation times to 15minutes also can be used. A 500 uL aliquot of the substrate solution at37° C. was then added and, following mixing, the absorbance wasdetermined for 3 minutes at 460 nm using a Model 8450A Hewlett-Packardspectrophotometer. The activity of the Streptokinase plasminogen wasexpressed as % normal by comparison to the control plasma results. Theresults of twelve plasma samples with values ranging from 40% to 140%normal compared well to the reference assay results. The correlationcoefficient for the compared data was, r=0.990, and the least squaresequation was calculated as y=0.956x+4.08.

Example IV Spectrophotometric Prothrombin Time with (Sar-Pro-Arg)₂-Rhodamine 110

The prothrombin time (PT) is used to monitor the extrinsic pathway ofblood coagulation. This test is performed by the addition ofthromboplastin to a plasma sample, resulting in the activation ofclotting factor proteases, with the ultimate formation of a fibrin clot.The rate of clot formation is measured and is directly related to theextrinsic clotting factor activites in the plasma sample. The syntheticsubstrate (Sar-Pro-Arg)₂ -Rhodamine 110 can be substituted in this testfor the natural substrate fibrinogen. The final extrinsic pathwayclotting factor, thrombin, is detected by the synthetic substrate.

The substrate was prepared in 0.05M ACES buffer, pH 7.5, at 40 uMconcentration. Thromboplastin was added to the buffered substrate, 0.05mL per 1.0 mL substrate, and the mixture warmed to 37° C. Twenty-five uLof a plasma sample was placed in a semi-micro acrylic cuvette, 1 cm.light path, Evergreen Scientific. One half milliliter of thethromboplastin substrate mixture was added to the cuvette containing theplasma sample and the change in absorbance at 496 nm measured with aModel 8450A Hewlett-Packard spectrophotometer. Synthetic substrate testresults for 56 patient samples, including some from patients receivingthe anticoagulants heparin and/or coumadin, which are antiactivators,were compared to prothrombin time (PT) clotting values. Three commercialthromboplastin reagents were used: General Diagnostics, OrthoDiagnostics, and Dade Diagnostics. The patient sample clotting timesranged from 9.8 to 40.6 seconds as determined by a Coag-a-Mate®2001,photo-optical clot detection system, General Diagnostics. The syntheticsubstrate test results were inversely related to the clotting assayvalues, and the correlation coefficients for the compared data using the3 thromboplastins were -0.944, -0.953 and -0.963.

Example V Spectrophotometric Antithrombin III-Heparin Cofactor Assaywith (Sar-Pro-Arg)₂ -Rhodium 110

Antithrombin III was measured in plasma samples using the water solublesubstrate, (Sar-Pro-Arg)₂ -Rhodamine 110, and the reference fluorescentsubstrate assay procedure of Mitchell, G. A. et al., Thrombosis Research12, 219-225 (1978) and the results were compared. Five uL of plasma and50 uL of purified water were added to a semi-micro disposablepolystyrene cuvette, 1 cm light path, Evergreen Scientific. Afterwarming to 37° C., a one-half milliliter aliquot of a solutioncontaining 5 units/mL of thrombin and 10 units/mL of heparin, inbuffered saline was added to the cuvette. The mixture was incubated at37° C. for 2 minutes 48 seconds. An activation time of 2 minutes 48seconds was used since it is the fixed time increment of the COULTERDACOS system. Five hundred uL of the water soluble substrate, at aconcentration of 375 ug/mL in 0.1M ACES, pH 7.0, with 0.05M KCl at 37°C. was added to the cuvette. The change in absorbance at 460 nm wasdetermined for 20 seconds. The results for 26 plasma samples withAntithrombin III levels ranging from 40 to 115% of normal correlatedwell with the reference assay values. The correlation coefficient forthe compared data was, r=0.983 an the least squares equation wascalculated as y=1.026x-2.90.

The novel water soluble xanthylium derivative substrate also can be usedto simultaneously monitor the extrinsic and intrinsic pathways ofthrombin formation by the well known thrombotest, a method firstdescribed by Dr. P. A. Owren, Lancet II, pp 754-758 (1959). Thethrombotest is sensitive to the factors of both the extrinsic andintrinsic pathways, and the thrombin generated from two pathways isapproximately equal. The thrombotest reagents include thromboplastin,partial thromboplastin, adsorbed plasma, and fibrinogen. The novel watersoluble xanthylium derivative substrates can be substituted in thethrombotest for the natural substrate, fibrinogen, to determine theactivity of the thrombin formed.

The above examples support the use of the water soluble rhodamine 110and rhodol derivative substrates in the universal coagulation tests, theprothrombin time, the activated partial thromboplastin and thethrombotest.

Although particular embodiments and examples of the invention have beenshown and described in full here, there is no intention to thereby limitthe invention to the details of such embodiments and examples. On thecontrary, the intention is to cover all modifications, alternatives,embodiments, usages and equivalents of the subject invention as fallwithin the spirit and scope of the invention, specification and theappended claims.

What is claimed is:
 1. A method to obtain a hydrolyzed product fordetermining the activity of a sample, said sample including at least aproteolytic enzyme; said sample may include one or more member of thegroup consisting of: an inhibitor, a cofactor, an activator, and anantiactivator, for said enzyme, said method comprising the stepsof:admixing a xanthylium derivative substrate with said sample, andpermitting the admixture to stand for a period of time sufficient toobtain a hydrolyzed product capable of being used for determining theactivity in said sample; said xanthylium derivative substrate beingselected from the group consisting of: ##STR3## and acid salts thereof,wherein: R₁ is a water solubilizing radical selected from the groupconsisting of sacrosyl, pyroglutamyl, and glutaryl; R₂ is xanthylium,3',6'-diamino-9'-(2-carboxyphenyl); and R₃ is xanthylium,3'-amino-6'-hydroxy-9'-(2-carboxyphenyl).
 2. The method as defined inclaim 1, wherein said sample includes an activator, said proteolyticenzyme includes plasminogen; and further including the step ofpreactivating said proteolytic enzyme before admixing said sample withsaid substrate.
 3. The method as defined in claim 1, wherein said sampleincludes an inhibitor and an antiactivator, and said inhibitor includesantithrombin III; and further including the step of preincubating saidsample with thrombin before admixing with said substrate.
 4. The methodas defined in claim 3, including the step of determining the activity ofsaid sample by at least one of fluorometric and spectrophotometricmeans.
 5. The method as defined in claim 1, including the step ofdetermining the activity of said sample.
 6. The method as defined inclaim 5, and further including the steps of treating said substrate withthromboplastin, and determining the activity of the formed thrombin. 7.The method as defined in claim 6, wherein said sample also includes atleast one member of the group consisting of: an inhibitor, a cofactor,an activator, and an antiactivator for said enzyme.
 8. The method asdefined in claim 5, in which said sample further includes a cofactor andan activator; and further including the steps of treating said substratewith activated partial thromboplastin, and determining the activity offormed thrombin.
 9. The method as defined in claim 5, in which saidsample further includes a cofactor and an activator; and furtherincluding the steps of treating said substrate with thromboplastin,partial thromboplastin and adsorbed plasma, and determining the thrombinactivity generated from both the extrinsic and intrinsic pathways. 10.The method as defined in claim 1, wherein R₁ is sarcosyl.
 11. The methodas defined in claim 1, wherein R₁ is pyroglutamyl.
 12. The method asdefined in claim 1, wherein R₁ is glutaryl.
 13. The method as defined inclaim 1, wherein said sample also includes at least one member of thegroup consisting of: an inhibitor, a cofactor, an activator, and anantiactivator for said enzyme.